PROJECT SUMMARY/ABSTRACT Cholestatic liver diseases are devastating illnesses of the biliary system, accounting for ~45% of pediatric and 10% of adult liver transplants annually. Currently no targeted therapies are available, and there is a significant unmet clinical need to improve patient survival. Biliary epithelial cells (BECs) possess significant regenerative capacity, but after severe cholestatic injury hepatocytes (liver parenchymal cells) are able to transdifferentiate into BECs to repair damaged bile ducts in both adult and pediatric models of disease. Hepatocytes are thus an untapped reservoir for promotion of biliary repair. Recently, Yes-associated protein 1 (Yap1) has emerged as a critical regulator of bile duct formation and liver regeneration. However, little is known about the mechanisms by which Yap1 drives biliary differentiation during embryonic development or hepatocyte-derived BEC regeneration. We have developed a novel Foxa3-Cre Yap1 knockout (KO) mouse model in which Yap1 is fully deleted from liver progenitor cells before induction of the biliary lineage, resulting in failure of bile duct formation and severe cholestatic injury. A recent model of Alagille syndrome showing similar defects in biliary development described extensive de novo generation of bile ducts derived from hepatocytes by 4 months. However, 4-month-old Yap1- deficient livers show no evidence of hepatocyte-derived biliary regeneration. Based on these observations, our overarching hypothesis is that Yap1 is indispensable for functional bile duct formation regardless of their origin, whether from hepatoblasts during embryonic liver development or from transdifferentiating hepatocytes in the setting of severe cholestatic injury. To investigate, we propose the following specific aims, which will yield novel mechanistic insights into Yap1 signaling, with significant implications for regenerative medicine and tissue engineering technologies aimed at promoting biliary repair and regeneration. Specific Aim 1: We will determine the ontogeny of Yap1 activity during normal liver organogenesis, and investigate the timeline of bile duct development in Yap1 KO mice using immunofluorescence and 3D whole-liver microscopy. We will thus identify the specific defects in 3D bile duct morphogenesis caused by Yap1 deletion. Specific Aim 2: Based on our preliminary data showing lack of hepatocyte-to-BEC transdifferentiation in Yap1 KO mice, we hypothesize that restoration of Yap1 expression to Yap1-negative hepatocytes will selectively allow these cells to transdifferentiate and restore a critical biliary mass to ameliorate ongoing cholestatic injury. We will use lineage- tracing to determine whether any hepatocyte-derived bile ducts are present in adult Yap1 KO mice. We will then use two complementary approaches to selectively deliver a tagged wild-type Yap1 construct to a small number of hepatocytes in Yap1 KO mice and trace their fate to assess their capacity for transdifferentiation into BECs and de novo bile duct formation. Contribution to Training: This proposal combines research training in liver pathology, developmental biology, and state-of-the-art biological imaging with world-class clinical training, providing a strong foundation for a productive career as an academic physician scientist.